GSM7049148: Lung tumor Flt3-YFP-positive macrophages; Mus musculus; RNA-Seq
Mice were sacrificed by cardiac perfusion with ~10-15mL PBS-heparin under isofluorane anesthesia. Liver, lung, or pancreas tissue was then isolated, minced with scissors, and digested in ~15-25mL Dulbecco's Modified Eagle Medium (DMEM) with 2mg/mL Collagenase A and 1x DNase I for 25 minutes (lung and liver tissue) at 37°C with constant stirring. Digestion buffer was quenched with 3mL fetal bovine serum (FBS) and filtered through 100uM Nylon mesh, pelleted by centrifugation (2000rmp for 5 min) and resuspended in FACS buffer (1% BSA in PBS). Cells were then resuspended in FACS Buffer containing CD16/CD32 antibodies to block for 10 minutes on ice, pelleted by centrifugation, then resuspended in fluorophore-conjugated antibodies, stained for 25 minutes on ice, then washed with FACS Buffer. Cells were then immediately sorted on Aria-II (BD Biosciences). After cells were sorted into FACS Buffer, they were pelleted by centrifugation, resuspended in 0.04% BSA in PBS, and cell counts were obtained. Single-cell RNA-sequencing library preparation was then conducted by the Genome Technology Access Center at Washington University. Briefly, cells from each sample were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3'v3 Reagent kits according to the manufacturer's protocol (10x Genomics, Pleasanton, CA, USA).